Shank Family Proteins

 Shank Family Proteins are scaffolding proteins found in the postsynaptic density (PSD) of excitatory synapses. The PSD, a structure within the postsynaptic membrane of dendritic spines, contains a complex assembly of proteins which organize neurotransmitter receptors and regulatory elements. The PSD coordinates communication of incoming signals to various targets and changes its composition in response to neural signals to aid neuronal plasticity Shank proteins function as the master organizer of the PSD with their ability to recruit and form multimeric complexes with postsynaptic receptors, signaling molecules, and cytoskeletal proteins, like AMPA, Neuroligin and NMDA glutamate receptors. Within the PSD, there are over 300 individual shank molecules, roughly 5% of the total protein molecules within the PSD. Shanks contain five domains for protein-protein interactions, including an ankyrin repeat domain, used to bind acting regulating proteins, an Src homology 3 (Sh3) domain, used to bind AMPA receptors, a PDZ domain, used to bind G protein coupled receptors, several proline-rich domains, and a C-terminal SAM domain, which is responsible for mediating Shank multimerization. (See Image) Shank also mediates the maturation of dendritic spines in neurons.

Chromosome 22q13 Deletion Syndrome
Chromosome 22q13 deletion syndrome (22q13DS) is a neurobehavioral syndrome marked by global developmental delay, and autism spectrum disorder (ASD) features. The Shank-3 gene is located within this region of chromosome 22. Studies have revealed that point mutations in Shank-3 can cause the neurodevelopmental symptoms associated with 22q13DS, accounting for 1% of all autism cases. At the molecular level, disruption of the full length Shank-3 protein reduces AMPA receptor signaling and spine remodeling. Mice who were haploinsufficient for Shank-3, emitted fewer ultrasonic vocalizations during interactions with estrus female mice, a behavior reminiscent of that seen in Autism patients. Further, Shank knockout mice have less dendritic spine development, a diminished PSD size, decreased levels of proteins GKAP and Homer, and greatly impaired synaptic signaling. Interestingly, overexpression of Shank-3 may also result in an ASD, supporting the hypothesis that Autism is caused by improper Excitatory/Inhibitory neuronal ratios in the brain. Measurements of broad miRNA expression levels in Autism patients uncovered aberrant levels of miRNAs for genes involved in ASDs like MeCP2, the cause of Rett Syndrome, NRXN-1, a gene implicated in ASDs, and Shank-3, adding support to Shank-3’s role in autism. Due to the marked reduction in AMPA receptor signalling in Shank-3 mutants, compounds that enhance AMPA transmission (AMPAkinses) serve as potential therapeutic approaches to treating some ASDs.

βPIX Structure
βPIX is a protein belonging to a group of guanine nucleotide exchange factors used by Rho GTPase family members, like Rac1 and Cdc42. Rac1 and Cdc42 regulate the actin cytoskeleton of synapses. PIX has an N-terminal Src homology 3 (SH3) domain which associates with PAK, a coiled-coil (CC) domain, which is critical for multimerization, and a C-terminal PDZ binding domain which interacts with the PDZ domain of Shank. The interaction of Shank with βPIX promotes the synaptic localization of βPIX and βPIX associated p21 Associated Kinase (PAK). Since PAK regulates actin cytoskeletons, and dendritic spines are actin-rich structures, it is believed that Shank recruits βPIX to dendritic spines to regulate the PSD.

Shank Family Protein Structure
The canonical PDZ domain contains 90 amino acids and folds into a compact globular structure consisting of a six-stranded β-sandwich flanked by two alpha helices. βPIX possess a parallel trimer via helical hydrophobic interactions within its CC domain, a proline to break the helix, and a PDZ binding domain at the C-terminus. Interestingly, only 1 Shank molecule is bound to the CC domain trimer of βPIX in an asymettric assembly. The 8-residue PDZ binding domain of βPIX forms a number of hydrogen bonding and hydrophobic interactions with the Shank PDZ domain. Shank-3-Arg 679 forms the most critical interaction with βPIX, tightly H-Bonding Glutamate 643, forming 2 weak bonds with Phe 696, and Van der Waals interactions with ring of Phe 696. Abolishing this interaction through mutagenesis completely eliminates the assembly. Upon binding of βPIX, the PDZ domain undergoes a significant conformational change. Lys 682 undergoes a nearly 11 Angstrom displacement to make room for the βPIX PDZ binding domain.

Shank Oligomerization
Shank proteins are positioned between scaffolding proteins that are bound to either neurotransmitter receptors or the actin cytoskeleton. This puts Shank proteins in a perfect position to create the underlying structure of the PSD. The SAM domain of Shank-3 canoligomerize (Alternate View ) to form large sheets composed of helical fibers stacked side by side. The proposed sheet structure with radially projecting protein interaction domains, is ideal architecture for a protein that must contact both membrane and cytoplasmic components at a synaptic surface. It resembles the structure of a peg board, with Shank oligomers forming the board and PIX proteins forming the pegs to which things attach. Models of this sort validate the importance of Shank-3 as master scaffolding proteins and illustrate how slight mutations can disrupt an entire PSD and synaptic function.

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Page Development
This article was developed based on lectures given in Chemistry 543 by Prof. Clarence E. Schutt at Princeton University.

Additional Structures of Shank Family Proteins
2fn3 - Crystal Structure of the native Shank SAM domain 2f44 - Crystal Structure of the Zinc-bound Shank SAM domain 1q3o, 1q3p - Crystal structure of the Shank PDZ-ligand complex